RESUMEN
The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Células Laberínticas de Soporte/metabolismo , Organogénesis/genética , Factor de Transcripción Brn-3C/genética , Factores de Transcripción/genética , Potenciales de Acción/fisiología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Calbindinas/genética , Calbindinas/metabolismo , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Proteínas de Homeodominio/metabolismo , Transporte Iónico , Células Laberínticas de Soporte/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Neuritas/metabolismo , Neuritas/ultraestructura , Parvalbúminas/genética , Parvalbúminas/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Transducción de Señal , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.
Asunto(s)
Transdiferenciación Celular/fisiología , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Transdiferenciación Celular/efectos de los fármacos , Transdiferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Células Ciliadas Auditivas/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Miosina VIIa , Miosinas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción Brn-3C/genética , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/genética , Tretinoina/farmacologíaRESUMEN
Transcription factors (TFs) have a central role to play in regulating gene expression. To analyze the co-expression patterns of selected TFs with the motor protein prestin of the outer hair cells, we applied an real-time PCR approach combining several kinds of information: (i) expression changes during postnatal development, (ii) expression changes by exposure of organotypic cultures of the organ of Corti to factors which significantly affect prestin expression [thyroid hormone (T4), retinoic acid (RA), butyric acid (BA), increased KCl concentration] and (iii) changes along the apical-basal gradient. We found that the mRNA levels of the TF Brn-3c (Pou4f3), a member of the POU family, are significantly associated with the regulation of prestin during postnatal development and in cultures supplemented with T4 (0.5 µM), BA (0.5-2.0 mM), and high KCl (50 mM) concentration. The mRNA level of the constitutively active TF C/ebpb (CCAAT/enhancer binding protein beta) correlates positively with the prestin expression during postnatal development and in cultures exposed to T4 and RA (50-100 µM). The mRNA levels of the calcium-dependent TF CaRF correlates significantly with the prestin expression in cultures exposed to T4 and high KCl concentration. The observed coexpression patterns may suggest that the TFs Brn-3c, C/ebpb, and Carf contribute to regulating the expression of prestin under the investigated conditions.